Molecular underpinnings of APTX Nicked DNA Sensing and Pleiotropic Inactivation in Neurodegenerative Disease The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder Ataxia with Oculomotor Ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. We reported X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes POLbeta and FEN1. Our results uncover a DNA induced-fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations. ZATT protein licenses removal of DNA-protein crosslinks: Topoisomerase 2 (TOP2) DNA transactions are essential for life, and proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. These TOP2 DNA protein crosslinks are also a potent form of cell killing DNA damage that is induced by widely used anticancer drugs, environmental toxicants, chemical metabolites, tobacco exposures, or DNA damage caused by ultraviolet light. How genotoxic TOP2 DNA-protein crosslinks are resolved is unclear. We reported discovery of a novel DNA damage response protein ZATT (aka ZNF451) that is critical for resolution of Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs). We show that the SUMO ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent Tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a split-SIM SUMO2 engagement platform. Our findings uncover a ZATTTDP2 catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc. Overall these results show how the ZATT-TDP2 complex is a novel sensor of TOP2-DPCs that facilitates the direct resolution of these highly genotoxic lesions, and underpins cellular resistance to TOP2 targeted cancer therapy. Development of Mammalian Protein expression tools: Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2 (340 KDa dimer) and a TOP2 catalytic core (260 KDa dimer). This robustly and reproducibly yields greater than 10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. The YFP affinity tag procedure described herein can be used with other means of delivering DNA, such as cationic lipid transfection reagents or engineered viral gene delivery, where conditions for transfection or infection may need to be optimized. Furthermore, since the anti-GFP/YFP nanobody is specific for these fluorescent proteins, co-expression with another tagged protein (i.e. monomeric red fluorescent protein) could allow for simultaneous detection of the individual proteins and optimization of expression levels. Altogether, the benefits of expressing YFP-tagged recombinant proteins overcomes key challenges of expressing recombinant proteins in human cells, and present a straightforward approach to generating recombinant proteins.